A number of considerations must be made in order to select a suitable system for over-expression of a desired gene product. Important issues include the yield of heterologous gene product required, costs of using the expression system and the authenticity/biological activity of the recombinant gene product produced in the production host.
As high-level production of a heterologous peptides, polypeptides or proteins will be a burden to the host cell, it may be advantageous to use an inducible, i.e. regulatable gene expression system that can be repressed during propagation of the production organism, both to avoid prolonged cultivation periods and to minimise the risk of selecting non-producing variants.
To develop an inducible expression system, which is suitable for production on an industrial scale, it is also important that induction of the system does not imply technical difficulties or the use of costly or toxic substances. Besides the quantity of product obtained from the production organism, the purity of the produced gene product is very important. A high yield of product contained in a whole-cell lysate may be reduced substantially during the subsequent steps of down-stream processing required to remove undesired host cell components. Therefore, secretion to either the periplasm in gram-negative bacteria or to the extracellular environment of gram-positive bacteria is generally preferred.
Until now Escherichia coli and Bacillus subtilis have been the most widely used bacterial host organisms for the recombinant production of peptides, polypeptides and proteins. The molecular biology of these organisms is characterised to a level that exceeds that for all other prokaryotic microorganisms and this extensive research has formed the basis for generating a large collection of genetic tools that have enabled easy cloning and expression of heterologous genes in these bacteria.
However, during the last decade there has been an increasing focus on the development of lactic acid bacteria (LAB) and in particular Lactococcus lactis as cell factories for production of homologous or heterologous peptides, polypeptides and proteins. LAB are advantageous for production of heterologous gene products in several aspects. The production and administration of recombinant peptides, polypeptides and proteins for pharmaceutical applications are subject to strict demands by regulatory authorities world-wide. For example, endotoxins, a component of the cell wall in most gram-negative bacteria, should be absent in the final product. Lactic acid bacteria do not produce endotoxins, which makes them attractive protein production host organisms. In addition, several lactic acid bacterial strains including L. lactis strains do not produce extracellular proteases and are capable of secreting peptides, polypeptides or proteins ensuring high gene product stability facilitating the subsequent purification hereof.
The design of a number of promising inducible gene expression systems for use in lactic acid bacteria (Kok, 1996; Kuipers et al., 1997; Djordjevic and Klaenhammer, 1998) has been achieved through studies focusing on the regulation of gene expression in L. lactis and their phages. Useful lactic acid bacterial expression systems include the NICE system (de Ruyter et al., 1996), which is based on genetic elements from a two-component system that controls the biosynthesis of the anti-microbial peptide nisin in L. lactis. Two other useful inducible expression systems are based on genetic elements from the L. lactis bacteriophages φ31 (O'Sullivan et al., 1996; Walker and Klaenhammer, 1998) and r1t (Nauta et al., 1997).
Promoterless reporter genes in transposons, integration vectors or plasmids (van der Vossen et al., 1987; Israelsen and Hansen, 1993; Sanders et al., 1998) have been used to identify inducible promoters in LAB. These promoters are induced by changes in the environment such as pH (Israelsen et al., 1995) and concentration of salt (Sanders et al., 1998).
Gene expression systems induced by metabolites produced by the host cell or by conditions naturally occurring during host cell growth are of industrial interest due to the low cost and food grade status of the inducing factor. Therefore, we have exploited inducible promoters including the pH inducible P170 and derivatives hereof as disclosed in the co-owned published international patent applications WO 94/16086 and WO 98/10079 in the development of a new gene expression system for use in L. lactis. The transcription from the P170 promoter is induced by low pH during the concomitant transition to stationary phase, ie the expression is repressed during exponential growth phase. In a recent study, the P170 promoter was characterised in detail and the original expression level was increased approximately 150-200 fold by genetic engineering, without affecting the regulation (Madsen et al., 1999).
Although attempts to achieve cost-effective levels of gene product using lactic acid bacterial host cells have been promising, there is, however, a continued industrial need to increase the productivity of such production systems. Additionally, there is a need to provide fermentation processes where the medium does not contain potentially hazardous components that can be a health risk to the end-user of the products. Examples of such undesired components include animal viruses and prions, the presence of which cannot be completely excluded in conventional fermentation media containing components of animal origin such as nitrogenous components. However, most presently used fermentation media are chemically undefined media containing such components of animal origin.
There is therefore a strong demand to provide methods for producing heterologous peptides, polypeptides or proteins which methods are absolutely safe and which permit the desired gene products to be provided at the same and preferably higher yields than do current production methods.
Current methods of producing heterologous gene products using lactic acid bacterial host cells are based on batch cultivation of the host cells in chemically undefined, nutrient rich media. The present invention encompasses the use of a chemically defined, ie synthetic medium is such production processes. It was found that the use of such media in a conventional batch process resulted in a yield of gene product that was significantly lower than that achieved in a conventional nutrient rich and chemically undefined medium. It was, however, found that this problem could be overcome by performing the production process as a continuous process or a fed-batch process.